Heterochromatin has been implicated in the regulation of a wide range of cellular processes including gene expression, chromosome segregation and cellular differentiation. The mechanisms involved in targeting heterochromatin to specific regions of the genome, however, are not fully understood. We gained insight into this problem when we found that double stranded RNA synthesized from heterochromatic centromeric sequences becomes rapidly processed by the RNAi machinery resulting in sequence specific targeting of heterochromatin to centromeres. The main goal of this proposal is to determine how specific regions of the genome become targeted for heterochromatin assembly. Our specific aims are to: 1) Identify the centromeric repeat sequences that are important for heterochromatin assembly. Although it is known that centromeric repeats are sufficient to recruit heterochromatin, precisely which of these centromeric sequences are important for heterochromatin assembly and how they function to recruit silencing is not clear. We will directly test this by assaying the ability of various centromeric repeat sequences to recruit heterochromatin assembly to an ectopic euchromatic site. 2) Determine whether RNA mediated heterochromatin formation normally occurs in cis or in trans. We will address whether the centromeric repeat sequences located at heterochromatic regions in fission yeast function to recruit heterochromatin assembly in cis or rather act as targets for heterochromatin targeting via silencing signals, such as centromeric small RNAs generated in trans. 3) Identify factors involved in initiation of heterochromatin assembly. In specific aim 3 we will use a combination of strategies to identify novel factors involved in initiation of heterochromatin assembly. It is important that heterochromatin be confined to discrete chromosomal regions since improper targeting of heterochromatin can lead to misregulation of gene expression and onset of human diseases such as cancer. Our studies may lead to the development of techniques that allow manipulation of heterochromatic domains which would be extremely useful not only as experimental tools to study gene function but also as potential treatments for human disease.